Others (19%)
Pseudogene (13.2%)
Antisense (16%) LincRNA
(51.7%) Protein coding
(82.5%)
LncRNA (17.5%)
0 0.5 1 1.5 2 2.5 3
Chromatin RNA lev
els
normalized to INT
1
(re
la
tiv
e to
si
Cn
trl)
Chromatin RNA
si Cntrl si Dicer si Drosha
b
RPKM
Chromat
in RNA-s
eq
1600
1600
1600 si Cntrl
si Drosha
si DGCR8
RPKM
Chromat
in RNA-s
eq
3000
3000
3000 si Cntrl
si Drosha
si DGCR8
MIR17HG
MIRLET7BHG
a
MIR181A1HG
RPKM
Chromatin RNA-seq
si Cntrl
si DGCR8 si Dicer -1200
-1200
-1200
Western blot
α: Dicer
α: Actin
-3000 -3000 -3000
RPKM
Chromatin RNA-seq
si Cntrl
si DGCR8 si Dicer
LINC00472
a
b
b
TSS plot of lncRNA genes harbouring miRNA TSS plot of protein coding genes harbouring miRNA
a
a
b
si Cntrl
si Drosha
si DGCR8 3500
3500
3500
MIR17HG-GPC5
a
1000
1000
1000 si Cntrl
si Drosha
si DGCR8
GPC5-Exon1
b
RPKM
Chromatin RNA-seq
RPKM
Chromatin RNA-seq
GPC5 CDS
Online Methods
PCR primers and siRNA sequences
See Supplementary Table 6.
Antibodies
Glypican5 (ab124886; Abcam), OGFRL1 (SC-137654; Santa Cruz), anti-Actin (A2103; Sigma), anti-Drosha (ab12286; Abcam), anti-DGCR8 (10996-1-AP; Proteintech), anti-CPSF73 (A301-090A; Bethyl), anti-α-Tubulin (T5168; Sigma), anti-H3 (ab1791; Abcam), anti-β-tubulin (ab6046; Abcam), anti-Dicer (13D6; Abcam).
Plasmid Constructs
Construction of βwt (formerly labeled HIVβ) has been described previously (Dye and Proudfoot, 1999). The pri-miR-122wt construct was made by insertion of a genomic PCR fragment generated using the primers Pri122qF/Pri122qR on Huh7 genomic DNA. The resulting ~8kb PCR fragment was ligated into a cloning vector prepared by long range PCR amplification of βwt with primers Open_BetaqR/B10 qF using Prime STAR HS DNA polymerase (Takara). The Quick change II XL site directed mutagenesis kit (Stratagene) was used to generate the various mutants using the following primer sets: primiR-122∆ (DELTA mirqF/DELTA mirqR); pA1mt (pA1MTqF/pA1MTqR).
Cell culture and transfection of siRNAand plasmids
RNA isolation and northern blot
RNA was isolated using TRIzol reagent (Ambion) according to the manufacturer's instructions. Total RNA from human liver was purchased from Agilent Technologies (Cat. No. 540017). Northern blotting was carried out using standard procedures on equal molar quantities of RNA. Membranes were probed with a random-primed 32
P-labelled DNA fragment corresponding to nt 3077-3707 (exon probe) and nt 1563-2005 (intron probe) of pri-miR-122 in Ultrahyb (Ambion). A fragment corresponding to nt 685-1171 of -actin was used as a loading control. For miRNA northern blots, the small RNA fraction was isolated by dissolving total RNA in 300μL of TE buffer with addition of equal amounts of PEG solution (20% PEG 8000, 2M NaCl). Samples were mixed and incubated for at least 30 min on ice, followed by centrifugation at 14,000g for
15min and isopropanol precipitation of the supernatant. Small RNA were run on a 18% polyacrylamide (19:1) urea gel and analyzed as described before54 using a 32
P-end-labeled oligonucleotide complementary to miR-122.
Reverse transcription and real-time qPCR analysis
Total RNA was treated with DNase I (Roche) and reverse-transcribed using SuperScript Reverse Transcriptase III (Invitrogen) and random primers (Invitrogen). Real-time quantitative PCR (qPCR) was performed with 2x Sensimix SYBR mastermix (Bioline) and analysed on a Corbett Research Rotor-Gene GG-3000 machine.
Nuclear-cytoplasmic fractionation
The procedure for isolating nuclear and cytoplasmic RNA has been described elsewhere55.
m7G cap selection
Capped nuclear RNA was immunoprecipitated using a mouse monoclonal antibody against the 5´-terminal m7G cap (cat.No.201001; SYSY Synaptic Systems) according
PolyA+ and polyA- RNA separation
DNase I-treated nuclear RNA was incubated with oligodT magnetic beads (Dynabeads mRNA purification kit, Invitrogen) to isolate either polyA+ RNA, which was bound to beads, or polyA- RNA, which was present in the flowthrough after incubation. OligodT magnetic bead selection was performed twice to ensure pure polyA+ or polyA- populations. The polyA- RNA population was further processed with the Ribo-Zero Magnetic Kit (Human/Mouse/Rat, Epicentre) to deplete most of the abundant ribosomal RNA.
In vitro polyA tailing and 3’ RACE
To detect the 3’ end of pri-miR-122, in vitro polyA tailing of nuclear RNA from Huh7
was carried out first using the polyA tailing kit (AM1350; Ambion) according to the manufacturer’s protocol. Purified RNA was reverse-transcribed using an oligodT24V
anchor using SuperScript III (Invitrogen). Compatible linker and sense primer (fwd1) were used for PCR. PCR fragments were gel purified and cloned into TA cloning vector (StrataClone PCR cloning kit) for sequence verification.
RNA stability
Transcript half-life was estimated after actinomycin-D (5mg/ml) treatment of Huh7 cells. Transcript levels at the indicated time points were analyzed by qPCR. The relative levels of expression of each transcript at the different time points were normalized according to the levels at time 0.
Western blot
Br-UTP nuclear run-on analysis
The Br-UTP NRO was carried out largely as described30 followed by qPCR analysis
as described above. The primers used are listed in Supplementary Table 6.
Chromatin RNA isolation
The procedures for separating nuclear RNA into chromatin-associated and released fractions have been described before56. Chromatin-associated RNA from Huh7 cells
was analyzed using qPCR as detailed above. The primers used are listed in Supplementary Table 6.
RNA-sequencing
Chromatin-associated RNA was isolated as described above with the omission of tRNA in solution preparation. Nuclear poly(A)- or poly(A)+ RNA were prepared as described above. RNA-seq was performed by the High‐Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics, University of Oxford. RNA Samples were ribodepleted using Ribo-Zero rRNA removal kit (Human/Mouse/Rat, EpiCentre RZH110424). Libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina, v1.0 (cat # E7420) using manufacturer's guidelines with an exception of using our own 8bp tags for indexing according to57. Libraries were
sequenced on an Illumina HiSeq-2000 using 100bp and 50bp paired end reads, v3 chemistry.
Bioinformatic analysis
Datasets
All human miRNAs and their genomic coordinates were obtained from miRBase release 2058. Annotation for protein coding genes (GRCh37) was obtained from
Ensembl, release 7459. Annotation for long noncoding RNA (lncRNA) was obtained
from GENCODE v7 catalog of human long noncoding RNA50.
considered for this study. This resulted in 112 lnc-pri-miRNA and 967 protein coding pri-miRNA. An additional 18 lnc-pri-miRNA were added to this list which mapped to genes having ‘lincRNA’ as Ensembl gene biotype and gene length ≥200 bp.
Genomic distribution of miRNA as belonging to protein coding genes and long non-coding genes was based on Ensembl gene biotype annotation (Supplementary Fig.1).
Mapping of sequencing reads
Paired-end reads for each sample were mapped to the human genome reference assembly GRh37/hg19 (build 37.2, Feb 2009) using the Bowtie2 alignment software60.
Prior to alignment, the first 12 nucleotides were trimmed from all the reads owing to the low quality of the bases. Uniquely mapped reads with no more than two mismatches were retained for further analysis. For nuclear polyA+ data, we filtered out reads that had 8 or more genomically encoded A-stretch at their 3’ ends. A statistical summary of read alignments can be found in Supplementary Table 5 for HeLa and Huh7 chromatin RNA-seq and HeLa nuclear polyA+ and polyA- RNA-seq.
Calculation of metagene profiles
We used the Ensembl gene annotation to define transcription start and end sites. In-house Perl and Python scripts were used to compute metagene profiles. To get a list of miRNA expressed in HeLa cells, miRNA FPKM was calculated using Cufflinks on small RNA-seq data for HeLa cells downloaded from the ENCODE Experiment Matrix available as ENCODE Project at UCSC61. This resulted in 15 lncRNA expressing 34
miRNA in HeLa cells. For the protein coding dataset, miRNA harboring genes with length ≥2 kb and the gene body RPKM ≥ 1 were considered. 545 genes were identified in this category.
To investigate the profile surrounding TSS upon Microprocessor depletion, we plotted normalized read count across a region of 1kb upstream and downstream of annotated TSS for si Cntrl, si Drosha and si DGCR8 in HeLa cells.
For supplementary Table 1 and 2, normalized read count (RPKM) was calculated over a region from the TES (3'end) of the lnc-pri-miRNA to 1 kb upstream of the TSS of the next downstream gene for miRNA harboring lncRNA genes that are expressed (RPKM≥1) in HeLa and Huh7 cell lines.
Classification of polyA+, polyA- and bimorphic transcripts
Classification of transcripts into polyA+, polyA- and bimorphic was done as described62. Briefly, all expressed transcripts were classified as polyA+, polyA- and
bimorphic predominant transcripts based on their relative abundance, calculated using BPKM (bases per kilobase of gene model per million mapped bases) in the polyA+ and polyA- sample for each condition (si Cntrl and si DGCR8). See Supplementary Table 3.
References
54. Pall, G.S. & Hamilton, A.J. Improved northern blot method for enhanced detection of small RNA. Nat. Protoc.3, 1077-84 (2008).
55. Dye, M.J., Gromak, N. & Proudfoot, N.J. Exon tethering in transcription by RNA polymerase II. Mol. Cell21, 849-59 (2006).
56. West, S., Proudfoot, N.J. & Dye, M.J. Molecular dissection of mammalian RNA polymerase II transcriptional termination. Mol. Cell29, 600-10 (2008).
57. Lamble, S. et al. Improved workflows for high throughput library preparation using the transposome-based nextera system. BMC Biotechnol. 13, 104
(2013).
58. Kozomara, A. & Griffiths-Jones, S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res.39, D152-7 (2014).
59. Flicek, P. et al. Ensembl 2014. Nucleic Acids Res.42, D749-55 (2014).
60. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat.
61. Kent, W.J. et al. The human genome browser at UCSC. Genome Res.12,
996-1006 (2002).
62. Yang, L., Duff, M.O., Graveley, B.R., Carmichael, G.G. & Chen, L.L. Genomewide characterization of non-polyadenylated RNAs. Genome Biol. 12,
Supplementary figure legends
Figure 1: Distribution of miRNA between lncRNA and protein coding genes.
17.5% of all miRNA derive from lncRNA. These can be further subdivided into intergenic (lincRNA) or other less well-characterized subdivisions such as pseudogene or antisense.
Figure 2: Transcript properties of lnc-pri-miR-122.
Sequencing results of lnc-pri-miR-122 3’RACE products amplified by poly(A) polymerase dependent method.
Figure 3: Drosha but not Dicer is required for lnc-pri-miRNA-122 transcription
termination.
Chromatin-associated RNA was analyzed by qPCR as in Fig. 3a. The efficiency of siRNA mediated knockdown of Drosha and Dicer is shown by western blots in Fig 6e and Supplementary Fig 5a respectively.
Figure 4: Chromatin RNA-seq profiles of two lnc-pri-miRNA in HeLa cells.
a. Chromatin RNA-seq profile showing that Drosha or DGCR8 depletion leads to
transcriptional readthrough at the MIR17HG locus. b. Chromatin RNA-seq profile showing no
transcriptional readthrough at the MIRLET7BHG locus following Drosha or DGCR8 depletion.
Figure 5: Dicer depletion does not lead to transcriptional readthrough on
lnc-pri-miRNA.
a. Western blot showing effective Dicer depletion by siRNA transfection in HeLa cells. b.
Figure 6: Effect of Microprocessor knockdown on levels of TSS transcripts harboring
miRNA in HeLa cells.
a. and b. TSS metagene plot of chromatin RNA-seq of lnc-pri-miRNA versus protein coding
genes harboring intronic miRNA showing region 1 kb before and after TSS.
Figure 7: Scatter plots showing reproducibility of replicate Chromatin RNA-seq.
a. si Cntrl (HeLa) b. si DGCR8 (HeLa).
Figure 8: Additional views of GPC5 Chromatin RNA-seq profiles.
a. Compressed view showing full extent of GPC5 transcription unit. b. Magnified view of GPC5
Supplementary Table 1:
List of miRNA harbouring lncRNA host genes showingtermination defect following Drosha/DGCR8 depletion in HeLa cell line.
RPKM fold increase >= 2 is considered as genes showing read-through.
Shaded region in the table highlights lncRNA host genes that do not show termination defect following Drosha/DGCR8 depletion.
lncRNA host gene miRNAs strand Normalized Read counts (RPKM) Fold increase Read-through
si Cntrl si Drosha si DGCR8 si Drosha si DGCR8 si Drosha si DGCR8
MIR17HG hsa-miR-(17~18a~19a~19b-1~20a~92a-1) (+) 57424.66 193251.73 339808.14 3.37 5.92 YES YES
MIR181A2HG hsa-miR-(181a-2~181b-2) (+) 450.27 2687.66 4790.68 5.97 10.64 YES YES
RP11-2B6.3 hsa-let-(7a-1~7f-1~7d) (+) 60.04 433.49 9390.42 7.22 156.41 YES YES
RP11-631N16.2 hsa-let-7i (+) 6211.77 13798.26 70840.17 2.22 11.40 YES YES
LINC00478 hsa-miR- (99a~let-7c~125b-2) (+) 1891.14 4306.04 6127.20 2.28 3.24 YES YES
MIR181A1HG hsa-miR-(181a-1~181b-1) (-) 630.38 82826.30 112737.08 131.39 178.84 YES YES
LOC646329 hsa-miR-(29a~29b-1) (-) 6393.86 148211.68 223252.40 23.18 34.92 YES YES
MIR31HG hsa-miR-31 (-) 2401.45 25821.81 26817.37 10.75 11.17 YES YES
LINC00472 hsa-miR-(30a~30c-2) (-) 14648.84 74459.86 114837.34 5.08 7.84 YES YES
MIR100HG hsa-miR-(100~125b-1~let-7a-2) (-) 4007.42 22772.90 31538.61 5.68 7.87 YES YES
DLEU2 hsa-miR-(15a~16-1) (-) 26145.78 93620.31 62868.93 3.58 2.40 YES YES
MIRLET7BHG hsa-let-(7a-3~7b) (+) 410.31 609.19 494.30 1.48 1.20 NO NO
LOC284454 hsa-miR-23a~24-2~27a (-) 2716.64 4739.54 5414.20 1.74 1.99 NO NO
MIR22HG hsa-miR-22 (-) 120.07 187.85 69.43 1.56 0.58 NO NO
Supplementary Table 2
: List of miRNA harbouring lncRNA host genes showing termination defect following DGCR8 depletion in Huh7 cell line.
RPKM fold increase >= 2 is considered as genes showing read-through.
Shaded region in the table highlights lncRNA host genes that do not show termination defect following DGCR8 depletion
Genes expressed in both HeLa and HUH7 cell lines are highlighted in bold.
*Based on our data: Read through calculated over chromosomal region (chr18:56120306-56130306) due to lack of available gene annotation.
lncRNA host gene miRNAs strand Normalized Read counts (RPKM) Fold increase Read-through si Cntrl si DGCR8 si DGCR8 si DGCR8 MIR17HG hsa-miR-(17~18a~19a~19b-1~20a~92a-1) (+) 16404.90 72189.92 4.40 YES
MIR181A2HG hsa-miR-(181a-2~181b-2) (+) 810.49 8748.19 10.79 YES
RP11-2B6.3 hsa-let-(7a-1~7f-1~7d) (+) 435.26 5901.55 13.56 YES
MIR146A hsa-miR-(146a~3142) (+) 1335.80 19336.27 14.48 YES
RP11-65J21.3 hsa-miR-(193b~365a) (+) 1650.99 7862.95 4.76 YES
RP11-631N16.2 hsa-let-7i (+) 3347.02 12445.34 3.72 YES
AP000662.4 hsa-miR-130a (+) 4012.84 9425.13 2.35 YES
lnc-pri-miR-122* hsa-miR-122 (+) 18556.80 41382.39 2.23 YES
MIR940 hsa-miR-(940~3677) (+) 60.04 121.50 2.02 YES
MIR181A1HG hsa-miR-(181a-1~181b-1) (-) 120.07 20915.81 174.19 YES
LOC646329 hsa-miR-(29a~29b-1) (-) 2671.61 63840.95 23.89 YES
AB429224 hsa-miR-194-2 (-) 285.17 2395.38 8.40 YES
MIR137HG hsa-miR-(137~2682) (-) 450.27 2655.70 5.89 YES
AC034220.3 hsa-miR-3936 (-) 120.07 624.87 5.20 YES
MIR210HG hsa-miR-210 (-) 300.18 1128.24 3.76 YES
DLEU2 hsa-miR-(15a~16-1) (-) 24629.87 83472.31 3.39 YES
LINC00472 hsa-miR-(30a~30c-2) (-) 645.39 1614.25 2.50 YES
MIRLET7BHG hsa-let-(7a-3~7b) (+) 14.65 22.43 1.53 NO
LINC00478 hsa-miR- (99a~let-7c~125b-2) (+) 420.25 833.16 1.98 NO
MIR22HG hsa-miR-22 (-) 255.15 433.93 1.70 NO
LOC284454 hsa-miR-23a~24-2~27a (-) 615.37 1041.45 1.69 NO
MIR600HG hsa-miR-600 (-) 2341.41 2655.70 1.13 NO
Shaded region in the table highlights lncRNA host genes that do not show termination defect following Drosha/DGCR8 depletion.
si Cntrl si DGCR8
BPKM BPKM Fold polyA BPKM BPKM Fold polyA lncRNA host gene pA(-) pA(+) change status pA(-) pA(+) change status
MIR17HG 21.37 1.89 11.31 pA(-) 43.69 5.8 7.53 pA(-) MIR181A2HG 3.24 0.49 6.59 pA(-) 4.25 1.03 4.12 pA(-) RP11-2B6.3 3.37 0.37 9.03 pA(-) 7.3 0.95 7.7 pA(-) RP11-631N16.2 6.12 1.03 5.91 pA(-) 13.35 2.66 5.02 pA(-) LINC00478 1.46 0.17 8.45 pA(-) 2.17 0.32 6.78 pA(-) MIR181A1HG 1.07 0.07 10.31 pA(-) 3.2 0.45 7.04 pA(-) LOC646329 2.02 0.5 4 pA(-) 10.6 2.96 3.58 pA(-) MIR31HG 4.18 0.51 8.12 pA(-) 6.95 1.22 5.68 pA(-) LINC00472 7.65 1.02 7.62 pA(-) 22.9 3.81 6.01 pA(-) MIR100HG 1.59 0.27 5.93 pA(-) 7.58 1.26 6.04 pA(-) DLEU2 2.27 0.58 3.93 pA(-) 3.2 0.97 3.28 pA(-) MIRLET7BHG 9.36 3.67 2.55 pA(-) 20.07 57.29 0.35 pA(+) LOC284454 15.49 46.46 0.33 pA(+) 19.96 69.89 0.29 pA(+) MIR22HG 6.05 1.58 3.82 pA(-) 9.82 7.27 1.35 bimorphic MIR600HG 1.41 1.12 1.26 bimorphic 1.73 1.38 1.26 bimorphic
Supplementary Table 3
: Polyadenylation status of miRNA-expressing lncRNAhost genes in control vs DGCR8 depleted HeLa cells.
Criteria for polyA status:
pA(-): Fold change values ≥2
pA(+): Fold change values ≤0.5
Supplementary Table 4
:Gene length and distance of pre-miRNA from TSS in lnc-pri-miRNA showing Microprocessor-mediated transcription termination in (a) HeLa and (b) Huh7 cells.a
b
c
lncRNA host gene Gene length Distance from TSSɫ
MIR17HG 6759 2785
MIR181A2HG 40164 33975
LINC00478 556874 468567
RP11-2B6.3 34338 9668
RP11-631N16.2 11323 934
LINC00472 76425 32616
LOC646329 39660 4255
MIR181A1HG 129936 51380
DLEU2 98587 21840
MIR31HG 104027 56473
MIR100HG 113959 10654
lncRNA host gene Gene length Distance from TSSɫ
MIR210HG 2797 2429
AB429224 4987 2893
MIR940 5263 2558
MIR17HG 6759 2785
RP11-631N16.2 11323 934
AP000662.4 14766 3174
MIR146A 19158 6134
RP11-65J21.3 24065 1679
RP11-2B6.3 34338 9668
LOC646329 39660 4255
MIR181A2HG 40164 33975
AC034220.3 58630 54204
MIR137HG 61863 57242
LINC00472 76425 32616
DLEU2 98587 21840
MIR181A1HG 129936 51380
ɫFor miRNAs in a cluster, distance is calculated from the TSS to the first miRNA
Sample ID RNA-seq Type Read size (nt)
Number of PE reads
Mapped PE reads
% of mapped PE reads
si_Cntrl Chromatin Hela 51 38343118 33313212 86.88%
si_DGCR8 Chromatin Hela 51 33569636 28805961 85.81%
si_Drosha Chromatin Hela 51 38989688 34602536 88.75%
si_Dicer Chromatin Hela 51 17923502 12819860 71.53%
si_Cntrl Chromatin Huh7 100 20678124 18305816 88.53%
si_DGCR8 Chromatin Huh7 100 25042908 21524505 85.95%
si_Cntrl_PolyAplus Nuclear Hela 100 22919390 15798545 68.93% si_Cntrl_PolyAminus Nuclear Hela 100 21002155 14045951 66.88% si_DGCR8_PolyAplus Nuclear Hela 100 24108556 16607260 68.89% si_DGCR8_PolyAminus Nuclear Hela 100 25086373 17768424 70.83%
SupplementaryTable 5
:RNA-seq mapping statistics.Supplementary Table 6:
Oligonucleotide primers and siRNAs used in studyPrimer (qRT-PCR) Gene Sequence (5’-3’)
EX1 forward lnc-pri-miR-122 CAGAAGCTGTGGAAGGCGC
EX2 reverse lnc-pri-miR-122 GAAACAGTGAGAGGTGAACAATTCA
INT1 forward lnc-pri-miR-122 TGCCACTCAGCACAGCACTA
INT1 reverse lnc-pri-miR-122 TGAACCCATCCTGCTCATA
GAPDH mRNA forward GAPDH AAGGTGAAGGTCGGAGTCAA
GAPDH mRNA reverse GAPDH AATGAAGGGGTCATTGATGG
U6 snRNA forward U6 snRNA CTCGCTTCGGCAGCACA
U6 snRNA reverse U6 snRNA AACGCTTCACGAATTTGCGT
EXON2 forward lnc-pri-miR-122 ACATGCCTGTGTCCACTGCT
EXON2 reverse lnc-pri-miR-122 CTCTAGGTGGCCCCAGTCAC
MIR+2.5kb forward lnc-pri-miR-122 GGACTGGGTCTTCCATGCTC
MIR+2.5kb reverse lnc-pri-miR-122 AAACGAATCCAGGCAGCAAT
MIR+4.5kb forward lnc-pri-miR-122 CATTCACTCCTGCACGCATT
MIR+4.5kb reverse lnc-pri-miR-122 GGTTCTGGGGAGAGCATCAC
MIR+7.5kb forward lnc-pri-miR-122 GCAACTGAAGCAGCATCGTT
MIR+7.5kb reverse lnc-pri-miR-122 CACGGGGGAGCTTTTCTCTT
INT1 forward GAPDH CCCCTTCATACCCTCACGTA
INT1 reverse GAPDH GACAAGCTTCCCGTTCTCAG
PAS forward GAPDH CTGAATCTCCCCTCCTCACA
PAS reverse GAPDH TGCCCCAGACCCTAGAATAA
PAS+1.1kb forward GAPDH TCCAGCCTAGGCAACAGAGT
PAS+1.1kb reverse GAPDH TGTGCACTTTGGTGTCACTG
Exon1 forward GPC5 TCGGCTAGGGAAGAAGACCA
Exon1 reverse GPC5 GCAGTTAGCTCTTCCCGGAG
-2kb GPC5 forward GPC5 TGACCTCCCACCTGTTTTGT
-2kb GPC5 reverse GPC5 AACGGTGTGTCTTGCAAGGA
exon1-2 forward GPC5 GAAACTTTTCCAGTGGCGGC
exon1-2 reverse GPC5 CTCCTCCATCTTCCTGGTGC
exon3-4 forward OGFRL1 TCCATTCAAGCCAGATGGTGT
exon3-4 reverse OGFRL1 AGTGTGGTTGTGCTCCAGTT
Primer (PCR-Gel) Gene Sequence (5’-3’)
exon2 (Total) forward lnc-pri-miR-122 GGTGAAGAGGTGAGAGTTGGA
exon2 (Total) reverse lnc-pri-miR-122 CAGCAAACCCCTTCTGCAAA
EX1 (spliced) forward lnc-pri-miR-122 CAGAAGCTGTGGAAGGCGC
EX2 (spliced) reverse lnc-pri-miR-122 GAAACAGTGAGAGGTGAACAATTCA
INT1 (unspliced) forward lnc-pri-miR-122 ACAACTGGGCTCAAGGGATC
INT1 (unspliced) reverse lnc-pri-miR-122 TCTGTTTAGCTTCCCTGGGC
GAPDH mRNA forward GAPDH AAGGTGAAGGTCGGAGTCAA
GAPDH mRNA reverse GAPDH AATGAAGGGGTCATTGATGG
fwd1 (3’end mapping) lnc-pri-miR-122 CCCGTGATGCTTCTTTTCTC
Linker (3’end mapping) GCTGTCAACGATACGCTACGTAACG
3’RACE-(dT)24V GCTGTCAACGATACGCTACGTAACG(T)24V
Anti-miR-122 (Northern blot)
AACACCAUUGUCACACUCCAUA (RNA oligo)
siRNA Gene siRNA Forward Strand
si Cntrl Luciferase GAUUAUGUCCGGUUAUGUAUU
si DGCR8 DGCR8 CAUCGGACAAGAGUGUGAU.dTdT
si Drosha Drosha CGAGUAGGCUUCGUGACUU.dTdT
si CPSF-73 CPSF3 ON-TARGETPLUS SMARTPOOL
CAT NO. L-006365-01
si Dicer Dicer UGCUUGAAGCAGCUCUGG.dTdT
Primers for pri-miR-122 cloning and mutagenesis
Sequence (5’-3’)
Open_Beta qR AGCTTTATTGAGGCTTAAGC
B10 qF CAGGAAACTATTACTCAAAGGG
Pri122 qF CAGAAGCTGTGGAAGGCGC
Pri122 qR ACTGTGTTCTCTGCTGCCTC
DELTA mir qF GTTTCCTTAGCAGAGCTGCAATCCTTCCCTCGATAA
DELTA mir qR TTATCGAGGGAAGGATTGCAGCTCTGCTAAGGAAAC
pA1MTqF CAATCTTGTGTACTTACTGAAGAAAGTCTGGCTCTTTTGCA
CT
pA1MTqR AGTGCAAAAGAGCCAGACTTTCTTCAGTAAGTACACAAGAT
TG
